Coagulopathies
Hemophilia A is a blood disease linked to the X chromosome, caused by deficiency or abnormality of factor VIII (FVIII), a cofactor necessary for the generation of fibrin. This deficiency of coagulation protein is the most common among coagulopathies, with an incidence of approximately 1 in 5,000 men and is currently affecting approximately 400,000 people worldwide. Hemophilia B is a hereditary disease that is also associated with the X chromosome and consists of the deficiency of blood coagulation factor IX, with an incidence of 1 in every 30,000 men. Clinically, both hemophilia A and B present many similarities, that is, the patient presents frequent bleeding episodes, most of the time in cutaneous, musculoskeletal and soft tissue regions. Bleeding can also occur in other critical spaces, such as, for example, intracranial or retroperitoneal space.
Conventional therapy for patients with hemophilia consists of intravenous infusion of factor VIII or FIX derived from plasma or recombinant protein. However, one of the major problems is the formation of inhibitory antibodies against FVIII and FIX, which is currently, the most significant treatment-related complication in the clinical attendance of hemophiliac patients. Approximately 5% of patients with hemophilia B and 20 to 30% of patients with severe hemophilia A, submitted to FIX and FVIII replacement therapy, respectively, develop antibodies that inhibit the activity of the infused factor. The treatments available for these patients include the use of hemostatic agents and the induction to immunologic tolerance using high doses of FVIII or FIX infusions. These approaches are expensive because of the high cost of the factors, and not always successful. For this reason, many efforts have been made in an attempt to find an hemonstatic effective treatment, independent of the presence of factor VIII and IX.
Over the years, many studies have identified activated factor VII (FVIIa) as an attractive candidate for hemostasis, regardless of the use of FVIII/FIX in animal with hemophilia models. In addition, the FVIIa purified from plasma, has been shown to induce hemostasis in some patients having severe hemophilia. Taken together, these data suggest that pharmacological doses of FVIIa bound to the tissue factor (TF) exposed in the injury site, activate FX and promote the formation of thrombin in the injury site, causing this coagulation factor to present itself as an alternative for hemophiliac patients with inhibitory antibodies.
Mechanisms of Action of FVII in Normal Haemostasis and the Role of Pharmacological Doses
According to the current concept, hemostasis occurs in two major types of surface: the cells that express tissue factor (TF) and platelets activated by thrombin and is initiated by the formation of a complex between the exposed TF and the FVIIa present in the circulation. FVII/FVIIa is the natural ligand of the tissue factor and the formed complex is fairly strong and stable.
Once the complex between TF and FVIIa is formed, the formation of a limited amount of thrombin occurs. This limited number of thrombin molecules formed in the initial phase of hemostasis activate the cofactors FVIII, FV, FXI and the platelets. Once activated, the platelets leave the circulation and go to the injury site. The activation of factors VIII and IX on the surface of activated platelets promotes activation of factor X in FXa, which in its turn binds to FVa generating a large amount of thrombin. The final step in the process is of a firm fibrin clot, which is resistant to premature proteolysis and is capable not only of initiating, but also of maintaining homeostasis, while the healing process is established.
In the absence of FVIII or FIX, only a small amount of thrombin is generated by the TF-FVIIa complex and the generation of total thrombin, which begins on the surface of platelets, does not occur. This last phase depends on the formation of the FVIII-FIX complex on the surface of the activated platelets. As a result, fibrin clots formed in hemophiliac patients are fragile and easily dissolved by premature proteolysis. From studies of hemophilia in cellular models, it was possible to demonstrate that pharmacological concentrations of recombinant factor VIIa (rFVII) bind non-specifically to activated platelets and generate thrombin on the surface thereof, even in the absence of FVIII/FIX. This occurs because rFVIIa activates FX on the surface of activated platelets independent of the presence of FVIII or FIX.
In this way, the addition of pharmacological doses of rFVIIa results in the rapid increase in the rate of thrombin generation on the activated platelet surface and as a result of increased activation of the platelets at the site of injury, increased adhesion platelets was observed, as well as other mechanisms necessary to maintain the homeostasis.
On Mar. 25, 1999 the FDA (Food and Drug Administration) approved the use of the first and only recombinant factor VII, NovoSeven®. Distributed by NovoNordisK, the recombinant activated factor VII (rFVIIa) is indicated in the treatment of bleeding episodes in patients having haemophilia A and B that develop antibodies against factors VIII and IX, respectively. In addition, rFVIIa is recommended for the treatment of critical spontaneous and/or surgical bleeding which threaten the lives of patients, as well as in patients with other diseases such as: FVII deficiency and Glanzmann's thrombasthenia.
Factor VII Gene
The factor VII gene has its locus located in region 34 of the long arm of chromosome 13 (13q34). Structurally and functionally, they are related to the group of vitamin K dependent serine proteases, which include factors IX, X, prothrombin (FII) and protein C. Its size is approximately 12.8 Kb and is composed of nine exons and eight introns. The nucleotide sequence of the exons is completely known. It is known that exons 1a and 1b and part of exon 2 encode a peptide signal that is removed during processing. The rest of the exon 2 and exons 3 to 8 encode a protein of 406 amino acids present in the blood circulation.
FVII is synthesized in the liver and circulates in the blood in a concentration of 0.5 μg/ml as a single chain, with a molecular weight of 50 kDa. In the amino-terminal moiety it consists of a domain rich in glutamic and γ-carboxylated acid (GLA domain), followed by two domains similar to epidermal growth factor (EGF), a short binding peptide and a serine protease domain in the carboxy-terminal moiety.
The conversion of factor VII to the active enzyme (FVIIa) occurs by the cleavage of the Arg152-Ile153 peptide bond, where no release of any peptide occurs. As a consequence, factor VIIa is composed of two polypeptide chains joined by disulfide bond. The light chain comprises the GLA domain, the aromatic helix and two EGF domains. This chain is composed of 152 amino acids that encode a protein of 20 kDa molecular weight. The heavy chain has the catalytic site of the molecule and comprises 254 amino acids with about 30 kDa molecular weight.
Factor VII and Vitamin K Dependent γ-Carboxylation
One of the main problems with the production of vitamin K-dependent recombinant coagulation factors for therapeutic use has been the deficient functional recovery of these proteins of the cell culture medium. Studies have shown that these results are mainly due to: 1) the incomplete γ-carboxylation of secreted proteins and 2) inefficient removal of the propeptide by Turin protease in the Golgi complex.
The vitamin K-dependent γ-carboxylation system is a system composed of several proteins located on the membrane of the endoplasmic reticulum. It consists of: 1) a vitamin K-dependent γ-carboxylase enzyme, which requires the reduced form of hydroquinone of vitamin. K (vit. K1H2) as cofactor and 2) the warfarin-sensitive enzyme, vitamin K 2,3-epoxide reductase (VKOR), which produces the cofactor. Concomitant with γ-carboxylation, hydroquinone is converted into the metabolite vitamin K 2,3 epoxide which is reduced back to the vit. K1H2 cofactor by the action of VKOR, in the so-called vitamin K cycle.
The calumeninee protein was identified as one of the factors capable of regulating the γ-carboxylation system, wherein the same would bind γ-carboxylase as an inhibitory chaperone and would also affect the VKOR protein. This conclusion is based on data that include: 1) the inhibition of γ-carboxylase activity with transfection of a construct containing the calumenine cDNA, 2) the silencing of the calumenine gene by a Smart siRNA and 3) a proteomic approach that demonstrates the existence of protein-protein interactions between β-carboxylase and calumenine. It has also been shown that when using Hek293 cells there was an increase in the production of recombinant FVII in these cells of 9% to 68%, when they were transfected for superexpression of the VKORC1 protein and concomitantly had the calumenine gene stably suppressed by more than 80% by the expression of a shRNA.
Within this context it is possible to predict that a human cell line has the proper machinery to γ-carbolixate and more efficiently produce recombinant FVII.